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Document Sample
PROTOCOLS FOR THE
HIGH-VOLUME ASSEMBLY
OF DNA BARCODES
Mehrdad Hajibabaei
Barcode of Life Initiative
University of Guelph
Canada
Barcoding Animals
Gaining Barcode Closure for Animals
10 million species
x
10 barcodes each
=
100 million barcodes
Barcoding Animals
Barcodes in Perspective
100 million barcodes
=
65 billion base pairs
Barcoding Animals
The Time Frame
10000
Time to Complete (Years)
1000
200 x 50K sequences per year
100
10 years to
completion
10
1
0.01 M
0.1 M
1M
10 M
100 M
Sequences/Year
Barcoding Animals
Logistical Challenges
How do we increase production rates,
lower costs and speed analysis?
How do we recover barcodes from
archival specimens?
The Barcoding Process
The Analytical Chain
Specimen or Tissue Sample
DNA Barcode
Extract DNA PCR COI Sequence COI
The Barcoding Process
Model 1: Core Facility
Specimen
Core Facility
50-100K Barcodes/Year
Specimen $1M Specimen
* Core Facility receives
specimen samples
Specimen
The Barcoding Process
Model 2: Satellite Labs
Satellite Lab $20K
Sequencing Facility
$800K
Satellite Lab $20K Satellite Lab $20K
Satellite Lab $20K * Sequencing Facility
receives PCR product
The Barcoding Process
The Guelph Analytical Node
Sourcing Specimens
Guelph Analytical Node Extracting DNA
DNA Amplification
Screening PCR Products
Sequencing
Barcode
of Life
Database
Sourcing Specimens
Sourcing Specimens
moderately
abundant
rare very
number of species
20 abundant
15
10
5
1 2 4 8 16 32 64 128 256 512
log abundance
Sourcing Specimens
Specimen Submission Package
DNA Extraction
DNA Extraction
DNA Extraction
Sample Material
Destructive tissue
sampling is the rule… But the amount is small
DNA Extraction
Non-Destructive Methods
Non-destructive methods are available
Minute specimens Type specimens
DNA Extraction
Two Favored Methods
Method Cost ($) Sensitivity
Chelex 0.10 +
Membrane 1.50 +++
DNA Amplification
DNA Amplification
Primer Design Optimized Reaction
DNA Amplification
Primer Design
100
75
PCR Success
50
25
0
DNA Amplification
Optimized Reaction
Removes the need for PCR
cleanup
Lower cost by decreasing
reaction volume
Screening PCR Products
Screening PCR Products
Screening PCR Products
A Fast-Gel Approach
Screening PCR Products
A Micro-Fluidic Approach
Screening PCR Products
A Comparison of Methods
Standard Fast-Gel Micro-fluidic
Agarose
Capital 0.5K 0.5K 100-250K
Maintence/Year N/A N/A 15-30K
Cost/Sample 0.03 0.3 0.5- 0.8
Time/96(min) 45 15 60
Sequencing
Sequencing
Sequencing Reaction Sequence Alignment
and Clean up
Sequencing
Sequencing Reaction and Clean up
1000
standard
reactions
$10,584
Sequencing
Sequence Alignment
Sequencher® SeqScape®
Data Management
The Need for a LIMS
NCBI
BoLD
Cost Analysis
Cost Analysis of Barcoding
Analysis Cost ($)
DNA Extraction 0.34
PCR 0.24
Fast-Gel 0.35
Sequencing X2 1.04
Cleanup X2 0.32
Sequence Run X2 0.40
Operational Costs 2.50
Total 5.19
Archived Specimens
Barcodes from Archived Specimens
Time
Barcode fragmentation
Archived Specimens
Full Barcodes versus Mini-Barcodes
Full Barcode Mini-Barcode
100
75
% Success
50
25
0
3-15 years 15-30 years
N=56 N=41
Archived Specimens
Immortalizing and Rescuing DNA
Whole genome
amplification
DNA repair
h
Archived Specimens
Whole Genome Amplification
1000-fold amplification of whole genome
Genomic DNA Amplified Genomic DNA Amplified Barcode
Region
Whole Genome Standard
Amplification PCR
Archived Specimens
DNA Repair
130 DNA repair genes in the human genome
DNA repair enzymes
Augmented PCR cocktails: i.e. Restorase®
Fragmented DNA Repaired DNA Amplified Barcode
Barcode Region
DNA repair Standard
protocol PCR
Archived Specimens
Early Restorase I Results
2002
Restorase I Regular Taq
100
% PCR Success
50
0
)
)
)
)
bp
bp
bp
bp
00
00
00
50 1930
(7
(6
(4
(3
1
2
3
4
R
R
R
R
PC
PC
PC
PC
Range of specimen
collection dates
Archived Specimens
Early Restorase II Results
2002
Restorase II Diamond Taq Regular Taq
100
% PCR Success
50
0
)
)
)
)
bp
bp
bp
bp
00
00
00
50
1930
(7
(6
(4
(3
1
2
3
4
R
R
R
R
PC
PC
PC
PC
Range of specimen
collection dates
Partnerships
Barcodes for Biodiversity Assessment
On the Horizon
On the Horizon
The Analytical Keychain
On the Horizon
On the Near Horizon
Speed – Specimen to
ID Time
barcode-based ID in
3 hours
Volume – 500K+ sequences
per-year via automation
Acknowledgments
Acknowledgements
Laboratory Database
Jeremy DeWaard Sujeevan Ratnasingham
Nataly Ivanova Rob Dooh
Stephanie Kirk
Janet Topan
Angela Hollis Paul Hebert Funding &
Support
Gordon and Betty Moore
Collections Foundation
Dan Janzen NSERC
John Burns
Canada Research Chairs
Jean-Francois Landry Program
